The current study demonstrated for the first time that a single episode of 60 cycles of mechanical stimuli can induce AC lesion in the lateral femoral condyle of rat, which is consistent with the results reported in smaller rodents like mouse [5–6]. The scope and localization of the lesion area were relatively steady in both the high and low load level groups as estimated by the chondrocyte-degenerative volume in each sample with low cartilage structural destruction except mild fibrillation in rare samples. However, the histological scores deteriorated over time due to the Safranin O staining loss and diffused hypocellularity in the affected area . ADAMTS-5, which is reportedly the primary catalyst for aggrecan degradation , was found to be overexpressed immediately after AC injury in the current study (Fig. 5). However, the Safranin O stained substances continued to degrade whereas the ADAMTS-5 expression dropped to normal levels after 4 weeks. Furthermore, the affected chondrocytes in the direct contact area did not demonstrate a positive response (Fig. 5). Studies on cell signaling have considered that overloading will activate the toll-like receptors expressed on chondrocytes, resulting in the release of proteinases and inflammatory cytokines . However, our results showed that color fading only occurred in the central region of cell death but not in the adjoining positive cells area. These results suggested that the aggrecanase of ADAMTS-5 might combine with other factors to augment proteoglycan degradation. We assumed that the integrity of fibrillar collagen networks or chondrocyte vitality may also play important roles in the maintenance of aggrecan homeostasis, which needs to be investigated in the future. Moreover, there was a distinct difference of staining between AC above and below the tidemark (Fig. 3A), which were consistent with the in vitro results that calcified radial zones of cartilage suffered less than 5% of the total mechanical stress [18–19].
The PTOA models of ACLT and DMM, which induced instability of the entire joint or deflection of load bearing towards medial or lateral tibial compartment, were widely used in rodents for short-term studies. The histological characteristics were defined as gradually developing chondrocyte apoptosis and cartilage matrix loss . However, we did not find any apoptotic cell around the lesion area even at very early observation of 6 h after loading (data not shown), which was considerably different from the previous results in mouse that illustrated that clustered active chondrocytes by TUNEL staining were retained in degenerative AC until 14 days after loading . Our results of live/dead staining demonstrated that chondrocytes in the superficial lesion cartilage were dead within 6 h by direct damage (Fig. 2). In this study, we set up the loading routine based on the weight ratio between rat and mouse as indicated in previous studies, which may have led to results slightly different from the previous study. We quantified the size of the lesion area and found that the volume containing degenerative chondrocytes had not changed much between each time point for both groups. However, the decreased Safranin O staining area enlarged over time that indicated the gradual depletion of glycosaminoglycans in the chondrocyte death region after loading. On the other hand, the AC shape destruction did not progress as rapidly as the invasive models, which illustrated jagged cartilage surface and subchondral bone perforation within 4 weeks after instability surgery [21–22]. According to our investigation, this non-surgical model may be better for the simulation of acute extensive AC damage, which is more common in the field of sport injuries.
Expression of Col2 was found to be transiently increased within 1 h after ex vivo mechanical loading in several experiments using extracted cartilage explants [23–24]. In the tissue engineering field, a recently published review  summarized biochemical anabolism of synthetic substrate-seeded chondrocytes subjected to in vitro dynamic loading, most of which demonstrated subsequent results of Col2 upregulation in response to varied loading regimens. Ragan PM  reported transient upregulation of type-II collagen within 4 h in extracted bovine cartilage explants subjected to static mechanical compression, but did not check the chondrocytes survival rate. The current study, to our knowledge, is a first report of focal enhanced staining of type II collagen on lesion cartilage which has undergone in vivo cyclic loading, even with the complete death of the affected surface chondrocytes within 6 h. A previous study reported decreased Safranin O and Col2 staining in an osteochondral defect model , which was directly created on the AC surface using a 1-mm biopsy punch. However, our model illustrated diametrically opposite Type II collagen response to cyclic loading. Although one of the major collagenases, MMP-13 overexpressed immediately after loading injury (Fig. 6), the morphological degradation of AC did not progress extensively as in regular OA development. Further studies should focus on whether type II collagen proliferation is beneficial or harmful to AC protection.
The lubricin localized in the cartilage surface is reportedly a protective and lubricating component of the O-linked glycoprotein . We found that superficial lubricin staining in the damaged area decreased drastically immediately after cyclic loading in comparison with the non-loaded region (Fig. 7). A decreased superficial cartilage lubricin/proteoglycan 4 level was confirmed in both in vivo  and ex vivo  experiments. Several studies have reported increased coefficient of friction within few hours of cyclic loading [30–31], and also that lubricin in the cartilage surface was denuded by loading, even in the joint where most chondrocytes remained alive throughout the observation period . Our study revealed similar results in a non-surgical model at early observation after in vivo cyclic compression, whereas the results of immunohistochemistry illustrated that cartilage surface lubricin staining diminished only in the lesion area where cell death occurred. Further studies should provide more quantitative data and explore if superficial lubricin depleted independently of the factor of cell death. On the other hand, in the experimental sheep [32–33] and horse [34–35] models, lubricin concentration in the synovial fluid was upregulated transiently at the acute phase after injury, and synovial PRG4 (The gene encodes Lubricin) expression presented positive correlation with TNFα and ADAMTS-5 . Our results similarly illustrated that cleavage products of MMP-13 and ADAMTS-5 mounted dramatically within 1 week after trauma and dropped to the basal level gradually (Fig. 5, 6). Further investigations should also focus on synovium response and check if in vivo cyclic loading will promote or inhibit the synovial cell secretion of intra-articular lubricin.
Meanwhile, after tracking different time points within 8 weeks, we found that superficial cartilage staining with lubricin gradually recovered to the normal level (Fig. 8). The results after 4 weeks indicated a different direction of OA progression compared to those with joint instability-induced OA. Although, lubricin expression on cartilage was found elevated in late-stage OA patients , the mainstream results on the posttraumatic OA of human  and animal joint instability models [28, 38–40] in a long-term observation demonstrated that the joint lubricin concentration decreased after injury or surgery. Combining the results of decreased Prg4 expression in unstable joint after forced movement [41–42] with our results, we hypothesized that the instability-induced persistent incentives should play a more important role than the magnitude of loading in determining irreversible lubricin loss. Several studies [39, 43–44] where exogenous recombinant lubricin was delivered to medial meniscectomized rats found that the cartilage surface protein was prevented from depletion in the experimental group. In the current study, we found a self-healing process in the loading-damaged AC without any lubricin supplementation, which indicates that endogenous lubricin is an important repair mechanism in the post-traumatic knee and provides a plausible explanation that the cartilage degradation progression of non-invasive loading model was slower than joint instability surgery models. Interestingly, although superficial cell death was confirmed completely within 6 h after loading, the locations of cartilage lacunae were strongly stained with lubricin even at 8 weeks after injury. Previous studies found chondrocytes encapsulated in agarose [19, 45] and cartilage explants  expressed higher prg4 gene subject to compressive strain, and the intensified lacunae staining around cells were confirmed in the immunofluorescence images , which is similar to our results. Further studies should focus on potential links between chondrocyte-derived lubricin and the mechanism of superficial cartilage repairment.
The current study has some limitations. First, it focused on investigating the mid-term changes over time from 3 days to 8 weeks after loading implementation. Since cell death, type II collagen biosynthesis and superficial lubricin degradation happened earlier than our expectation, further studies should more precisely design the unit of observation intervals in hours to disclose the full process of dynamic loading-induced cellular reaction. Second, we did not examine the synovial changes in response to loading. As we described above, upregulated inflammatory cytokines and PRG4 expression were found in multiple researches, which possibly induced the changes of microenvironment in the joint cavity, and weakened the significance of the antigen detection results in the AC. For instance, the results like the increase of cartilage surface-adhesive protective proteins like lubricin could be confused by the higher secretion of lubricin in synovial fluid after injury, which gives rise to the question whether lubricin recovery could ascribe to self-healing capabilities or is simply due to high concentration of lubricin in the environment. Study in the future should evaluate the synovium using quantitative techniques to eliminate internal interference factors. Third, in the current study, we only assessed the cartilage lesion on lateral femur condyle. The cartilage damage on the other contact surface of lateral tibia should be examined in the future. Last, we failed to compare the current model to any surgery-induced model. As we described above, the surface lubricin reportedly diminished in many injury-induced OA animals [28, 38–40], whereas it is still unknown if the factor of joint instability independently affected the progression of post-traumatic OA, especially in the lesion area. Further studies should combine invasive destabilization surgery to pre-existing lesion caused by cyclic compression, which could reflect the spatiotemporal changes of cartilage in the non-contact area.
In conclusion, we found a specific-localized AC lesion in both the 20 N and 50 N groups that underwent 60 cycles of compression in rat knee joints. The size of lesion was affected by the load level and the intensity of histological staining weakened with time after loading. The local expression of type II collagen was raised after repeated loading whereas lubricin in the cartilage surface was lost in response to cyclic compression. However, the distribution of superficial lubricin recovered at 4 weeks after non-surgical injury (Fig. 8). These results indicated that dynamic loading exceeding 20 N could damage the lateral femoral condyle AC in rat. Although the damage caused localized chondrocyte deaths and upregulated expression of degrading enzymes, endogenous repairments in well-structured joint worked by rebuilding the layer of protective proteins on the superficial cartilage.